The goal of this Phase I proposal is the development of a simple, cost-effective, and high-throughput method for direct and quantitative RNA detection. In molecular medicine, such a method would greatly facilitate the analysis of RNA viruses and the measurement of specific gene expression, currently pressing problems in the field. Despite this need, few techniques have emerged that are truly direct and quantitative over a broad range of concentration differences, while being readily adaptable to any genetic system. This proposal describes the development of a novel enzymatic assay for direct and quantitative RNA analysis in a microtiter plate-based format. This assay relies on structure-specific cleavage of oligonucleotides hybridized to the target RNA and can be used to detect any sequence. A combination of protein engineering and solid-phase capture approaches will be used to attain a sensitivity of detection of subattomole quantities of RNA without intervening conversion to DNA. The expected outcome of Phase I is an assay for quantitative monitoring of gene expression in 1 mg or less of tissue that will be adaptable to any RNA sequence. This system will be adaptable to a variety of low-cost readout platforms, including chemiluminescent, fluorescent and colorimetric detection. PROPOSED COMMERCIAL APPLICATION: NOT AVAILABLE